RNA-seq analysis, targeted long-read sequencing and in silico prediction to unravel pathogenic intronic events and complicated splicing abnormalities in dystrophinopathy.

Dystrophinopathy is caused by alterations in DMD. Approximately 1% of patients remain genetically undiagnosed, because intronic variations are not detected by standard methods. Here, we combined laboratory and in silico analyses to identify disease-causing genomic variants in genetically undiagnosed patients and determine the regulatory mechanisms underlying abnormal DMD transcript generation. DMD transcripts from 20 genetically undiagnosed dystrophinopathy patients in whom no exon variants were identified, despite dystrophin deficiency on muscle biopsy, were analyzed by transcriptome sequencing. Genome sequencing captured intronic variants and their effects were interpreted using in silico tools. Targeted long-read sequencing was applied in cases with suspected structural genomic abnormalities. Abnormal DMD transcripts were detected in 19 of 20 cases; Exonization of intronic sequences in 15 cases, exon skipping in one case, aberrantly spliced and polyadenylated transcripts in two cases and transcription termination in one case. Intronic single nucleotide variants, chromosomal rearrangements and nucleotide repeat expansion were identified in DMD gene as pathogenic causes of transcript alteration. Our combined analysis approach successfully identified pathogenic events. Detection of diseasing-causing mechanisms in DMD transcripts could inform the therapeutic options for patients with dystrophinopathy.

Independent predictors of neuronal adaptation in human primary visual cortex measured with high-gamma activity.

Neuronal adaptation is defined as a reduced neural response to a repeated stimulus and can be demonstrated by reduced augmentation of event-related gamma activity. Several studies reported that variance in the degree of gamma augmentation could be explained by pre-stimulus low-frequency oscillations. Here, we measured the spatio-temporal characteristics of visually-driven amplitude modulations in human primary visual cortex using intracranial electrocorticography. We determined if inter-stimulus intervals or pre-stimulus oscillations independently predicted local neuronal adaptation measured with amplitude changes of high-gamma activity at 80-150 Hz. Participants were given repetitive photic stimuli with a flash duration of 20 µs in each block; the inter-stimulus interval was set constant within each block but different (0.2, 0.5, 1.0 or 2.0s) across blocks. Stimuli elicited augmentation of high-gamma activity in the occipital cortex at about 30 to 90 ms, and high-gamma augmentation was most prominent in the medial occipital region. High-gamma augmentation was subsequently followed by lingering beta augmentation at 20-30 Hz and high-gamma attenuation. Neuronal adaptation was demonstrated as a gradual reduction of high-gamma augmentation over trials. Multivariate analysis demonstrated that a larger number of prior stimuli, shorter inter-stimulus interval, and pre-stimulus high-gamma attenuation independently predicted a reduced high-gamma augmentation in a given trial, while pre-stimulus beta amplitude or delta phase had no significant predictive value. Association between pre-stimulus high-gamma attenuation and a reduced neural response suggests that high-gamma attenuation represents a refractory period. The local effects of pre-stimulus beta augmentation and delta phase on neuronal adaptation may be modest in primary visual cortex.

Spontaneous and visually driven high-frequency oscillations in the occipital cortex: intracranial recording in epileptic patients.

High-frequency oscillations (HFOs) at ≥80 Hz of nonepileptic nature spontaneously emerge from human cerebral cortex. In 10 patients with extraoccipital lobe epilepsy, we compared the spectral-spatial characteristics of HFOs spontaneously arising from the nonepileptic occipital cortex with those of HFOs driven by a visual task as well as epileptogenic HFOs arising from the extraoccipital seizure focus. We identified spontaneous HFOs at ≥80 Hz with a mean duration of 330 ms intermittently emerging from the occipital cortex during interictal slow-wave sleep. The spectral frequency band of spontaneous occipital HFOs was similar to that of visually driven HFOs. Spontaneous occipital HFOs were spatially sparse and confined to smaller areas, whereas visually driven HFOs involved the larger areas including the more rostral sites. Neither spectral frequency band nor amplitude of spontaneous occipital HFOs significantly differed from those of epileptogenic HFOs. Spontaneous occipital HFOs were strongly locked to the phase of delta activity, but the strength of δ-phase coupling decayed from 1 to 3 Hz. Conversely, epileptogenic extraoccipital HFOs were locked to the phase of delta activity about equally in the range from 1 to 3 Hz. The occipital cortex spontaneously generates physiological HFOs which may stand out on electrocorticography traces as prominently as pathological HFOs arising from elsewhere; this observation should be taken into consideration during presurgical evaluation. Coupling of spontaneous delta and HFOs may increase the understanding of significance of δ-oscillations during slow-wave sleep. Further studies are warranted to determine whether δ-phase coupling distinguishes physiological from pathological HFOs or simply differs across anatomical locations.

Occipital gamma-oscillations modulated during eye movement tasks: simultaneous eye tracking and electrocorticography recording in epileptic patients.

We determined the spatio-temporal dynamics of cortical gamma-oscillations modulated during eye movement tasks, using simultaneous eye tracking and intracranial electrocorticography (ECoG) recording. Patients with focal epilepsy were instructed to follow a target moving intermittently and unpredictably from one place to another either in an instantaneous or smooth fashion during extraoperative ECoG recording. Target motion elicited augmentation of gamma-oscillations in the lateral, inferior and polar occipital regions in addition to portions of parietal and frontal regions; subsequent voluntary eye movements elicited gamma-augmentation in the medial occipital region. Such occipital gamma-augmentations could not be explained by contaminations of ocular or myogenic artifacts. The degree of gamma-augmentation was generally larger during saccade compared to pursuit trials, while a portion of the polar occipital region showed pursuit-preferential gamma-augmentations. In addition to the aforementioned eye movement task, patients were asked to read a single word popping up on the screen. Gamma-augmentation was elicited in widespread occipital regions following word presentation, while gamma-augmentation in the anterior portion of the medial occipital region was elicited by an involuntary saccade following word presentation rather than word presentation itself. Gamma-augmentation in the lateral, inferior and polar occipital regions can be explained by increased attention to a moving target, whereas gamma-augmentation in the anterior-medial occipital region may be elicited by images in the peripheral field realigned following saccades. In functional studies comparing brain activation between two tasks, eye movement patterns during tasks may need to be considered as confounding factors.

Electrocorticographic correlates of cognitive control in a Stroop task-intracranial recording in epileptic patients.

The human brain executes cognitive control, such as selection of relevant information in the presence of competing irrelevant information, and cognitive control is essential for us to yield a series of optimal behaviors in our daily life. This study assessed electrocorticographic γ-oscillations elicited by cognitive control in the context of the Stroop color-naming paradigm, with a temporal resolution of 10 msec and spatial resolution of 1 cm. Subjects were instructed to overtly read a color word printed in an incongruent color in the reading task, and to overtly name the ink color of a color word printed in an incongruent color in the Stroop color-naming task. The latter task specifically elicited larger γ-augmentations in the dorsolateral-premotor, dorsolateral-prefrontal and supplementary motor areas with considerable inter-subject spatial variability. Such Stroop color-naming-specific γ-augmentations occurred 500 to 200 msec prior to overt responses. Electrical stimulation of the sites showing Stroop color-naming-specific γ-augmentations resulted in temporary naming impairment more frequently than that of the remaining sites. This study has provided direct evidence that a critical process of cognitive control in the context of Stroop color-naming paradigm consists of recruitment of neurons essential for naming located in variable portions of the dorsolateral premotor and prefrontal areas.

Ictal high-frequency oscillations at 80-200 Hz coupled with delta phase in epileptic spasms.

Previous studies of epileptic spasms reported that ictal events were associated with high-frequency oscillations (HFOs) or delta waves involving widespread regions. We determined whether ictal HFOs at 80-200 Hz were coupled with a phase of slow-wave, whether ictal slow-waves were diffusely or locally synchronous signals, and whether the mode of coupling between HFOs and slow-wave phases differed between ictal and interictal states. We studied 11 children who underwent extraoperative electrocorticography (ECoG) recording. The phases and amplitudes of slow-waves were measured at the peak of ictal and interictal HFOs in the seizure-onset sites. Ictal HFOs were locked tightly to the phase of slow-wave at ≤1 Hz. Ictal slow-waves propagated from the seizure-onset site to other regions. In contrast, interictal HFOs in the seizure-onset site were loosely locked to the phase of slow-wave at ≤1 Hz but tightly to that of ≥3-Hz. Ictal slow-waves coupled with HFOs can be explained as near-field and locally synchronized potentials generated by the neocortex rather than far-field potentials generated by subcortical structures. Ictal slow-waves in epileptic spasms may be generated by a mechanism different from what generates interictal HFOs-slow-wave complexes.

Statistical mapping of ictal high-frequency oscillations in epileptic spasms.

PURPOSE: We assessed 636 epileptic spasms seen in 11 children (median 44 spasms per child) and determined the spatial and temporal characteristics of ictal high-frequency oscillations (HFOs) in relation to the onset of spasms. METHODS: Electrocorticography (ECoG) signals were sampled from 104-148 cortical sites per child, and the dynamic changes of ictal HFOs were animated on each individual's three-dimensional (3D) magnetic resonance (MR) image surface. KEY FINDINGS: Visual assessment of ictal ECoG recordings revealed that each spasm event was characterized by augmentation of HFOs. Time-frequency analysis demonstrated that ictal augmentation of HFOs at 80-200 Hz was most prominent and generally preceded those at 210-300 Hz and at 70 Hz and slower. Recruitment of HFOs in the rolandic cortex preceded the clinical onset objectively visualized as electromyographic deflection. The presence or absence of ictal motor symptoms was related more to the amplitude of HFOs in the Rolandic cortex than in the seizure-onset zone. In a substantial proportion of epileptic spasms, seizure termination began at the seizure-onset zone and propagated to the surrounding areas; we referred to this observation as the "ictal doughnut phenomenon." Univariate analysis suggested that complete resection of the sites showing the earliest augmentation of ictal HFOs was associated with a good surgical outcome. SIGNIFICANCE: Recruitment of HFOs at 80-200 Hz in the rolandic area may play a role in determining seizure semiology in epileptic spasms. Our study using macroelectrodes demonstrated that ictal HFOs at 80-200 Hz preceded those at 210-300 Hz.

Cortical gamma-oscillations modulated by auditory-motor tasks-intracranial recording in patients with epilepsy.

Human activities often involve hand-motor responses following external auditory-verbal commands. It has been believed that hand movements are predominantly driven by the contralateral primary sensorimotor cortex, whereas auditory-verbal information is processed in both superior temporal gyri. It remains unknown whether cortical activation in the superior temporal gyrus during an auditory-motor task is affected by laterality of hand-motor responses. Here, event-related γ-oscillations were intracranially recorded as quantitative measures of cortical activation; we determined how cortical structures were activated by auditory-cued movement using each hand in 15 patients with focal epilepsy. Auditory-verbal stimuli elicited augmentation of γ-oscillations in a posterior portion of the superior temporal gyrus, whereas hand-motor responses elicited γ-augmentation in the pre- and postcentral gyri. The magnitudes of such γ-augmentation in the superior temporal, precentral, and postcentral gyri were significantly larger when the hand contralateral to the recorded hemisphere was required to be used for motor responses, compared with when the ipsilateral hand was. The superior temporal gyrus in each hemisphere might play a greater pivotal role when the contralateral hand needs to be used for motor responses, compared with when the ipsilateral hand does.

Cortical gamma-oscillations modulated by visuomotor tasks: Intracranial recording in patients with epilepsy.

We determined how visuomotor tasks modulated gamma-oscillations on electrocorticography in epileptic patients who underwent epilepsy surgery. Each visual-cue consisted of either a sentence or hand gesture instructing the subject to press or not to press the button. Regardless of the recorded hemisphere, viewing sentence and gesture cues elicited gamma-augmentation sequentially in the lateral-polar occipital and inferior occipital-temporal areas; subsequently, button-press movement elicited gamma-augmentation in the Rolandic area. The magnitudes of gamma-augmentation in the Rolandic and inferior occipital-temporal areas were larger when the hand contralateral to the recorded hemisphere was used for motor responses. A double dissociation was found in the left inferior occipital-temporal cortex in one subject; the lateral portion had greater gamma-augmentation elicited by a sentence-cue, whereas the medial portion had greater gamma-augmentation elicited by a gesture-cue. The present study has increased our understanding of the physiology of the human visuomotor system.

Cortical gamma oscillations modulated by word association tasks: intracranial recording.

Recent studies have suggested that cortical activation can be measured using event-related augmentation of gamma oscillations in humans. We determined how commonly and differentially gamma oscillations (50-150Hz) were modulated by three distinct word-association tasks during extraoperative electrocorticography monitoring in a patient with focal epilepsy who underwent epilepsy surgery. He was auditorily presented names of common foods (e.g., apple) during each task. He was instructed to overtly verbalize the color (e.g., red) of each given food during the first association task, the taste (e.g., sweet) during the second task, and the texture (e.g., crunchy) during the third task. All three word-association tasks commonly elicited significant augmentation of gamma oscillations in the superior temporal gyrus, the middle temporal gyrus, and the inferior frontal gyrus, as well as the pre- and postcentral gyri. The food-texture association task specifically elicited significant gamma augmentation in the supramarginal gyrus. This preliminary study generated the hypothesis that word-association tasks may supplement functional language mapping using electrical stimulation. Differential gamma augmentation in the supramarginal gyrus might be attributed to a larger workload required in the food-texture association task compared with the remaining two tasks.

Factors influencing EPA+DHA levels in red blood cells in Japan.

The blood eicosapentaenoic and docosahexaenoic acid (EPA+DHA) concentration is an important inverse risk factor for sudden cardiac death. However, it is not known what kinds of factors influence the EPA+DHA levels in the total phospholipid fraction in red blood cells (RBC EPA+DHA) in Japan, who regularly eat more fish with increasing age. Four hundred and fifty-six healthy individuals (320 men and 136 women, 18 to 70 years old) were recruited between 2002 and 2005. RBC EPA+DHA were measured by gas chromatography and questionnaires were administered. Multivariate analysis indicated that there were significant correlations between RBC EPA+DHA and (i) dietary EPA+DHA (beta=0.31), (ii) age (beta=0.33), (iii) gender (beta=-0.15) and (iv) physical activity (beta=-0.11) but not with body mass index or smoking.

[Early and serial electrodiagnostic findings in childhood Guillain-Barré syndrome].

We investigated the usefulness of electrodiagnostic (EDX) studies for the early diagnosis of childhood Guillain-Barré syndrome (GBS). We retrospectively reviewed 5 patients (ages, 17-96 months) who fulfilled the diagnostic criteria of GBS. The EDX studies were performed at least twice;they included 1 or more following:motor nerve conduction study (MNCS), F-wave study, electromyography (EMG), and sensory nerve conduction study. The first and second EDX studies were performed at 8 days (range:4-13 days) and 14 days (range: 12-27 days) after the onset of motor symptoms, respectively. Although only 3 of 5 patients showed abnormal findings in the first MNCS, additional EDX studies, namely, F-wave study and EMG confirmed the presence of peripheral neuropathy in all patients. Regarding the classification of GBS subtypes, the results of the first EDX studies lead to the diagnosis of acute inflammatory demyelinating polyneuropathy (AIDP) in 3 patients and the remaining two were diagnosed with AIDP based on results of the second studies. We concluded that serial EDX studies, including F-wave studies, are essential for the early and definite diagnosis of childhood GBS.

HHV-6 encephalopathy with cluster of convulsions during eruptive stage.

The features of a characteristic type of encephalopathy caused by human herpesvirus-6 are presented. All patients have clusters of brief convulsions during the stage of skin eruptions. Sometimes the encephalopathy resulted in hemiplegia or mental retardation. Because the clinical course is similar, we propose a subtype of encephalopathy "human herpesvirus-6 encephalopathy with cluster of convulsions in eruptive stage" for further studies of pathophysiology and treatment.

[A patient with HHV-6 encephalopathy showing a cluster of convulsions during the eruptive stage and hyperperfusion of the cerebral hemisphere during the acute phase].

We report a case of "HHV-6 encephalopathy with cluster of convulsions during eruptive stage" (HECCES) that showed hyperperfusion in the cerebral hemisphere by single photon emission computed tomography (SPECT) during the acute phase. There are several reports that described brain hypoperfusion of HECCES by SPECT, but all of these descriptions involved the chronic phase. In contrast, our patient showed hyperpefusion on the day of seizure cluster. Since the prognosis for HECCES is sometimes poor, it is important to clarify the etiology and to develop a treatment. The description of this case is significant for these points.

Is vaccenic acid (18:1t n-7) associated with an increased incidence of hip fracture? An explanation for the calcium paradox.

High calcium intake may increase hip fracture (HF) incidence. This phenomenon, known as the calcium paradox, might be explained by vaccenic acid (18:1t n-7, VA), the highly specific trans fatty acid (TFA) present in dairy products. First, we ecologically investigated the relationship between 18:1 TFA intake and HF incidence using data from 12 to 13 European countries collected before 2000; then we measured the effects of VA and elaidic acid (18:1t n-9, EA) on osteoblasts from goldfish scales (tissues very similar to mammalian bone), with alkaline phosphatase as a marker; and finally we measured the effect of VA on mRNA expression in the scales for the major bone proteins type I collagen and osteocalcin. HF incidence was significantly correlated with 18:1 TFA intake in men (r=0.57) and women (r=0.65). Incubation with 1µmol/L VA and EA for 48h significantly decreased alkaline phosphatase activity by 25% and 21%, respectively. Incubation of scales with 10µmol/L VA for 48h significantly decreased mRNA expression for type I collagen and osteocalcin (by about 50%). In conclusion, VA may be causatively related to HF and could explain the calcium paradox. It may be prudent to reduce 18:1 TFA intake, irrespective of trans positions, to prevent HF.

Congenital monomelic neurogenic disorder with calf muscle hypertrophy.

An 8-year-old girl had hypertrophy of the right calf muscle since birth, with progressive ankle contracture and mild muscle weakness. Her right leg was 3 cm shorter than her left. Electromyography and biopsy of the affected muscle showed neurogenic changes. She also had neurogenic bladder and spina bifida occulta at the S1 level. We believe that the spina bifida was responsible for the neurogenic changes in her right calf, but could not find definite evidence for this association. Although cases with neurogenic muscle hypertrophy, especially calf muscle hypertrophy, have been reported, none of them was congenital or associated with spina bifida occulta.

γ-oscillations modulated by picture naming and word reading: intracranial recording in epileptic patients.

OBJECTIVE: We measured cortical gamma-oscillations in response to visual-language tasks consisting of picture naming and word reading in an effort to better understand human visual-language pathways. METHODS: We studied six patients with focal epilepsy who underwent extraoperative electrocorticography (ECoG) recording. Patients were asked to overtly name images presented sequentially in the picture naming task and to overtly read written words in the reading task. RESULTS: Both tasks commonly elicited gamma-augmentation (maximally at 80-100 Hz) on ECoG in the occipital, inferior-occipital-temporal and inferior-Rolandic areas, bilaterally. Picture naming, compared to reading task, elicited greater gamma-augmentation in portions of pre-motor areas as well as occipital and inferior-occipital-temporal areas, bilaterally. In contrast, word reading elicited greater gamma-augmentation in portions of bilateral occipital, left occipital-temporal and left superior-posterior-parietal areas. Gamma-attenuation was elicited by both tasks in portions of posterior cingulate and ventral premotor-prefrontal areas bilaterally. The number of letters in a presented word was positively correlated to the degree of gamma-augmentation in the medial occipital areas. CONCLUSIONS: Gamma-augmentation measured on ECoG identified cortical areas commonly and differentially involved in picture naming and reading tasks. Longer words may activate the primary visual cortex for the more peripheral field. SIGNIFICANCE: The present study increases our understanding of the visual-language pathways.

The effect of docosahexaenoic acid on aggression in elderly Thai subjects--a placebo-controlled double-blind study.

Docosahexaenoic acid (DHA) administration controls extraggression (aggression against others) in young subjects under psychological stress. However, it is not known whether its administration affects extraggression of elderly subjects. Forty Thai subjects of 50-60 years of age (22 males and 18 females) were recruited from Silpakorn University and nearby villages. They were allocated to the control and DHA groups in a double-blind fashion, and took 10 mixed plant oil capsules and 10 DHA capsules (1.5g DHA/day) for 2 months, respectively. Extraggression was measured with a psychological test (PF Study) at the beginning and end of the study. Just prior to the PF Study at the end of the study, subjects were asked to watch a stressful videotape as a stressor component. The average DHA intake from food was 150-160mg/day. In the group of university employees, extraggression did not change over time with placebo, whereas extraggression significantly decreased (31 +/- 13 to 24 +/- 13%, P = 0.04 by the paired-t test, P = 0.04 by ANOVA). In the group of villagers, there was no significant difference between the control and DHA groups in extraggression. The DHA administration favorably controlled extraggression in at least elderly white-collar workers. The daily intake of 150-160 mg/day of DHA was not enough to control extraggression.